Mucorales fungi for use in preparation of foodstuffs

ABSTRACT

The preparation of a proteinaceous substance suitable for use in a foodstuff is described which comprises fungal fells of the order  Mucorales . The cells are grown in a fermentor vessel in a liquid which is mixed during fermentation, after which the RNA content of the fungal cells is reduced to below 4% the fungal cells processed into an edible substance. This substance is then mechanically texturized into edible textured product for inclusion into foodstuffs, for example in the form of chunks as a meat substitute.

FIELD OF THE INVENTION

The present invention relates to the preparation of edible(proteinaceous) substances using fungal cells of the order Mucorales andthe use of these substances in foodstuffs, in particular as meatsubstitutes.

INTRODUCTION

Animal meat is considered to be a desirable part of the human diet, notonly due to the vitamins and nutrients it provides, but also due to itsflavour (particularly on cooking) and, importantly, its texture.However, an increasing number of people are turning to vegetarian orvegan diets, neither of which can include meat or meat derived products.Such diets may be due to a number of factors, but is often due to eithera disliking for meat (either in texture or flavour) or due to ethicaland moral considerations (for example, a belief that it is wrong to killanimals in order to feed humans).

The move towards vegetarian/vegan diets has increased in recent years bythe appearance of BSE (Bovine Spongiform Encephalopathy), otherwiseknown as “mad cow disease”, a disease that effects the nervous system incows and is thought to be as a result of feeding cattle parts of sheepinfected with a similar disease known as “scrapie”. BSE has been linkedwith a condition in humans known as CJD (Creutzfeldt-Jacob disease).

Apart from certain edible fungi (e.g. mushrooms) proteinaceous foodscontaining fungi are known. One example is the traditional Indonesianfermented food, tempeh. This is usually prepared by the fermentation ofRhizopus fungi on soy beans (and parts thereof) acting as a moist solidsubstrate. The beans (or other vegetable substrate) are inoculated withthe fungus and fermentation allowed for 24 to 36 hours. The beans becomebound by the fungal mycelium protein produced to give a firm productwhich can then be sliced before eating (no additional processing isusually performed before consumption). Thus the fungi are used tohydrolyse an otherwise inedible substrate and, apart from inherentlylacking much taste or flavour, tempeh is relatively dry and does nothave the fibrous and juicy texture associated with meat. The fungirepresent only a small amount of the product and so the fungal proteincontent is low. It is thus not particularly appealing as a meatsubstitute, at least for Westerners.

A number of edible meat substitutes or meat replacers have been proposedin recent years. Soy-based products, in particular extruded soy, aremarketed, especially by American and Japanese companies, but these donot have a particularly meat-like taste or texture (indeed both soy andgluten can both have an “off” or astringent taste).

GB-A-2007077 (Maclennan/BioEnterprises) proposes a similar process tothe manufacture of tempeh, except instead of soy beans the solidsubstrate is a starch-containing food such as sago, cereals or potatoes.However a prerequisite of this foodstuff (and tempeh) is that solidfoods or ingredients are needed as the substrate for the fermentingmicroorganisms.

More recently workers have proposed the production of edibleprotein-containing substances using the production of mycelial proteinby the fungus Fusarium graminearum. These substances have beenincreasingly used as meat substitutes, and are included in foodstuffssold in the UK and other European countries.

BRIEF DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a processfor the preparation of an edible (e.g. proteinaceous) substance,suitable for use in a foodstuff, comprising fungal cells, the processcomprising:

-   -   a. fermenting fungal cells of the order Mucorales in an aqueous        liquid contained in a fermenter vessel, the liquid comprising an        assimilable nitrogen (N) source and an assimilable carbon (C)        source, and mixing (and preferably aerating) the liquid and        cells during fermentation;    -   b. reducing the RNA content of the fungal cells;    -   c. before or after (b), removing at least some of the water from        the mixture of fungal cells and aqueous liquid; and    -   d. processing the fungal cells into an edible substance.

By using (non-toxic) Mucorales fungi (e.g. those used in Asian fermentedfood products) one can avoid any mycotoxins that may be produced byother (e.g. prior art) organisms. Thus little or no screening fororganisms that are safe for inclusion into foodstuffs may be required.This means that the products produced by the invention are more suitablefor ingestion and for use in foodstuffs. The Mucorales organisms ingeneral do not produce mycotoxins (or an insignificant amount or belowdetection), which is clearly advantageous as these organisms areincorporated whole into a foodstuff, and can mean that the processingtechniques can be more efficient as mycotoxins may not need to beremoved. This can overcome a problem with Fusarium organisms which canproduce undesirable mycotoxins (Desjardins et al, MicrobiologicalReviews, 57(3): 595–604, September 1993).

Mycotoxins anticipated here are those such as aflatoxine, mevinolin,terrein and trichothecenes (the latter being produced by some Fusariumspecies). A fungus that produces any of these mycotoxins is unlikely tobe allowed to be used in any form of food production, even if themanufacturer takes steps to remove the mycotoxins. It is thereforeparticularly important to choose fungi that will not produce thesemycotoxins at any stage of the process.

A further advantage of using Mucorales fungi is that a relatively widevariety of microorganisms is available, depending upon thecharacteristics desired in the proteinaceous substance. This can allowdiffering physical characteristics (such as in the fibrous nature, ormouthfeel) or in chemical characteristics (taste, etc). The fungi usedin the present invention have been found to give improved meat-likeproperties, for example a more fibrous and/or juicy texture. These fungican also vary in terms of texture and juiciness and so allow themselvesto be used in a wide variety of foodstuffs. Therefore by choice ofmicroorganism one can provide the various desirable characteristicsaccording to the eventual foodstuff to be prepared (using differentprocessing techniques). In addition different microorganisms can impartdifferent colours, and so as well as being able to prepare a whitesubstance, one can make substances that have different colours, such asyellow to brown or even green appearance, which may be desirable forsome foodstuffs.

The fungi can be of the family Choanephoraceae, such as of the genusBlakeslea or Gilbertella, for example of the species Blakeslea trisporaor Gilbertella persicaria. The other three families included within theorder Mucorales are Cunninghamellaceae, Mortierellaceae (such as fungiof the genus Morrierella, and in particular the species Mortierellaalpina) and, especially, Mucoraceae. Suitable fungi are usually edible(and digestible) by humans or animals.

Preferred fungi are saprophytic (that is to say, simple fungi) ratherthan parasitic (which are more complex). The “simple” fungi are usuallypreferred because they are better adapted towards hyphal growth, whereasthe parasitic organisms concentrate on taking nutrients from their“host” organism.

The fungal cells are preferably of the genus Rhizopus, Rhizomucor, Mucoror Mortierella, all of which belong to the family Mucoraceae. Suitablefungi of the genus Rhizopus, Mucor or Rhizomucor include Rhizopusstolonifer, Rhizopus miehei, Rhizopus pusillus, Rhizopus oligosporusand, in particular, Rhizopus oryzae, Mucor hiemalis and Mucor rouxii;and Rhizomucor meihei. Other preferred strains include those of thegenus Absidia or Phycomyces, such as Absidia pseudocylindrospora orPhyzcomyces blakesleeanus.

Preferred fungi can have a cell wall comprising, or primarilycontaining, chitin and chitosan. The cell walls may contain one or moreof the sugars glucosamine (such as D-glucosamine) and/or fucose, such asL-fucose, and may be substantially free of galactose.

The fungi used in the present invention preferably do not have septa,which is in contrast to those of the group Fusarium. Furthermore,preferred fungi for use in the invention have branching, again unlikeFusarium organisms which have little or no branching (in their hyphae).Indeed, the art advocates the use of non-branching mutants(EP-A-0,123,434). The hyphae of the fungi used in the invention may havea diameter from 1 to 20 μm, such as from 2 to 10 μm, optimally from 2 to8 μm.

The fungus may be a naturally occurring one, it may have been selectedusing known techniques for particular desired properties, or it may begenetically engineered.

Fungi of the order Mucorales are generally also of the group perfecti(in other words, not belonging to the class imperfecti) will beemployed, which are able to reproduce sexually. Fungi used in theinvention can also be filamentous.

As will be appreciated, the process of the first aspect is a liquidfermentation, in other words the aqueous liquid (e.g. a solution) servesas the culture medium. This contrasts with prior art processes whichculture fungi on a solid substrate, that substrate being, for example,rice, soy bean or starch-containing products such as cereals orpotatoes. In the invention the liquid fermentation process in (a) ispreferably conducted in the absence of a solid substrate, such as onewhich is itself an edible foodstuff (this includes not only vegetablematerial or legumes but also meat and natural solid starch orcarbohydrate-containing substances such as cereals, soybeans, sesameseeds or meal).

Thus a second aspect of the present invention relates to an edible (e.g.proteinaceous) substance, suitable for use in a foodstuff, comprisingfungal cells of the order Mucorales having a reduced (or low) RNAcontent. This substance may be preparable by a process of the firstaspect. It may be biomass or a filter cake, or such a biomass or filtercake which has been milled and/or tumbled.

A third aspect of the present invention relates to a process for thepreparation of an edible (textured) product, the process comprisingadding one or more edible components to an edible proteinaceoussubstance comprising fungal cells of the order Mucorales having areduced RNA content and (if necessary) texturising the mixture.

A third aspect also includes a process for the preparation of an edible(textured) product, the process comprising mixing one or more ediblecomponent(s) with an edible proteinaceous substance comprising fungalcells of the order Mucorales and texturising to form a product of whichat least 5% is fungal cells on a dry matter weight basis.

The fungal cells will preferably remain intact (or whole) not onlyduring the fermentation process, but also during subsequent processingsteps, including water removal, reduction of RNA content and anytexturing. Thus the substance (or textured product) will contain intact(but dead or killed) fungal cells, and during most if not all stages ofthe preparation of the substance or the product, steps will be taken tominimize damage to and lysis of the cells or cell membranes. However,during processing some compounds may leave the cell (so that the cellmembrane may “leak” a little). It is of course intended that the processof the invention involves the removal of some RNA (and if necessary alsowater) from the fungal cells.

The substance (and so also the textured product) contains fungal proteinproduced by the fungal cells. Usually this protein is intracellularand/or within the cell membrane. Extracellular protein may be presentbut these are often removed during processing (e.g. water removal, sincethe extracellular protein may be present in the aqueous liquid). Thusthe proteinaceous substance, for example a biomass, can be one that ispreparable by the process of the first aspect.

The proteinaceous substance preferably has at least 40%, e.g. at least50% or even 60% or more of fungal cells. However these amounts can bemuch larger and the cells can constitute at least 70%, such as at least80%, and optimally at least 90% or 95% of the proteinaceous substance(on a dry matter weight basis). With such a high content of fungal cellsone can obtain a substance (and, later also a textured product) that ismore juicy, fibrous and better tasting. Even after texturing theresulting edible (e.g. textured) product can have a fungal cell contentthe same as that quoted for the proteinaceous substance. Thesepercentages are based on the weight of the cells in the dry matter, inother words one firstly dries the substance or product and, on the basisof the dry matter obtained, calculate (by weight) the percentage of thatmatter that is fungal cells.

The substance can therefore consist essentially only of the fungal cells(which can include the protein produced by (e.g. inside) those cells).During fermentation, therefore, there will usually be no extraction ofisolation of any particular compound(s) or substance(s) either containedin or produced by the fungi as the fungal cells. Indeed in the processesof the invention it is preferable that RNA (or any degradation productsthereof or any compounds undesirable during processing) will be the onlycompound(s) that is removed from the cells. Extracellular protein may bewashed away from the cells and so may not be present.

The proteinaceous substance of the third aspect and textured product ofthe fourth aspect are edible in the sense that they can be included intoa foodstuff or they are compatible with food or feed use. Although theproteinaceous substance can be eaten as such, the intention is that thisis in fact an intermediate in the preparation of the textured product.

The proteinaceous substance may have, as the only edible material, thefungal cells and so may, apart from the fungal cells, be devoid ofedible substances. However, various edible component(s) can then bemixed with or added to the proteinaceous substance of the third aspectto produce the textured product of the fourth aspect. Further oradditional edible component(s) may be added to the textured product inthe preparation of the foodstuff.

A fourth aspect of the present invention is thus the textured product.This is suitable for inclusion into a foodstuff (it can be edible, andsuitably digestible, by either humans or animals) and can comprise cellsof the order Mucorales either having a reduced RNA content or where atleast 40% of the product (on a dry matter weight basis) is the fungalcells. The percentage of the fungal cells (on a dry matter weight basis)can be the same as that mentioned above for the proteinaceous substance(of the second aspect). However, as the product of the fourth aspect canbe prepared by adding edible component(s) to the proteinaceous substanceof the second aspect, it will be realized that the fungal cell contentof the former is often lower than that of the latter. The texturedproduct may comprise pellets, granules or sheets, it may be prepared bya process involving extrusion (and therefore may be an extrudate), maycomprise a dough, a paste, or a meat-like chunk, or may be in the formof a roll (such as by rolling the substance if it is in the form of asheet).

A fifth aspect of the present invention relates to a process for thepreparation of the foodstuff, which comprises forming a foodstuff with,or including into an existing foodstuff, an edible substance accordingto the second aspect (for example preparable by a process of the firstaspect) or a textured product of the fourth aspect (such as preparableby a process of the third aspect). This may involve adding one or moreadditional edible components to either the substance or to the product,or it may comprise further texturization. This process thereforeincludes not only preparation of a foodstuff using either the substanceor the product, but it also envisages the supplementation of a foodstuffwith either substance or product.

A sixth aspect of the present invention is thus provided by thefoodstuff. This can comprise either the substance of the second aspector the product of the fourth aspect. The foodstuff is preparable byprocess of the fifth aspect.

The foodstuff may be a sausage, pâté, burger, spread, animal feed or itmay include edible pharmaceutical compositions such as tablets.

The foodstuff preferably comprises at least 5%, for example at least 8or 10%, and optimally at least 15 or 20% of fungal cells (on a drymatter weight basis). The fungal content may be as high as thatdescribed for the textured product, but since the foodstuff can be madefrom that product the fungal content may be lower, for example thefungal cells may constitute only at least 25 or 30% of the foodstuff(again, based on a dry matter weight calculation).

DETAILED DESCRIPTION OF THE INVENTION

The fermentation process of the first aspect is suitably conducted in afermenter vessel adapted for containing the aqueous liquid, such as avat, and this vessel may be pressurized. It may be also be adapted toallow the continuous or continual supply of the assimilable nitrogenand/or carbon sources. Stage (a) and later stages are thereforepreferably conducted aseptically. Although the fermentation can be acontinuous process, with regular harvesting or removal of the fungalcells (and accompanying protein), the process can be a batch process ifdesired, such as a repeat fed batch process (one or more additions of Cand/or sources after fermentation has begun). Thus the fermentationprocess can be stopped or halted, and the fungal cells removed from thevessel, before another or fresh fermentation is begun.

The vessel may additionally be adapted to perform, or allow to beconducted, aeration and/or mixing of the cells and liquid, such asagitation of the solution, which may be stirring, for example achievedusing mechanical means.

The carbon and nitrogen sources may be provided in separatecompositions. This because the different sources may be subject todifferent sterilizing conditions, and furthermore it allows a variationin the relative amounts of carbon and nitrogen during fermentation.

The nitrogen and/or carbon sources can be supplied (or added)separately, or supplied simultaneously, or supplied as a combinedpreparation. They may thus present in the same composition (if thoughtnecessary) which is preferably a liquid. The C and/or N sources can beadded (to the fermenter vessel) either before the fungal cells are added(to the vessel), in other words prior to inoculation, or duringfermentation.

If the supply is continual (or intermittent), it is preferred that foreach instance of supply (e.g. “shots” or additions) the addition of bothcarbon and/or nitrogen sources is the same.

Preferred C:N (weight) ratios are at least 6:1, but may vary from 10:1to 150:1, such as from 15:1 to 50:1, optimally from 25:1 to 40:1.

For continual supply, preferably the time during which the nitrogenand/or carbon sources are supplied are greater than the time when theyare not. Thus, during fermentation supply is advantageous for at least50% of the time. If supply of one or both sources is intermittent, thenthere should be at least 2, preferably at least 5, and optimally atleast 10, additions to the aqueous liquid of the nitrogen and/or carbonsource. For continuous supply or further additions it is preferred thatthe C:N ratio in the sources is kept at (about) the same ratio as whenfermentation started.

The carbon and/or nitrogen sources may be complex sources, or individualor isolated compounds. Non-complex sources are preferred (these may haveor produce fewer mycotoxins) and so in the latter two cases these may beadded in a high degree of purity, and can be common (or commerciallyavailable) chemicals. Preferably both C and/or N sources are not solid,and suitably both are liquids.

Suitable nitrogen sources include ammonia or ammonium ions. Theadvantage here is that ammonia can act as a pH regulant. This may besupplied in the form of an ammonium salt, such as nitrate, sulphate orphosphate or in the form of ammonium ions themselves, for example anaqueous solution of ammonium hydroxide.

Other inorganic nitrogen sources can also be used, such as sodiumnitrate, urea or an amino acid such as asparagine or glutamine.

Other complex sources include yeast hydrolysates, primary yeast, soybean meal, hydrolysates of casein, yeast, yeast extract or rice bran.

The carbon source can comprise (complex sources such as) maltodextrin,oat flour, oat meal, molasses, vegetable (e.g. soy bean) oil, maltextract or starch. Preferred (non-complex) carbon sources includecarbohydrates or sugars, such as fructose, maltose, sucrose, xylose,mannitol, glucose, lactose, citrate, acetate, glycerol or ethanol.

Preferred nitrogen and/or carbon sources are water soluble or watermiscible.

The aqueous liquid may additionally contain other substances to assistin the fermentation, for example a chelating agent (e.g. citric acid),an anti-foaming agent (e.g. soy bean oil), a vitamin (e.g. thiamineand/or riboflavin), any necessary catalytic metals (for example, alkaliearth metals such as magnesium or calcium, or zinc or iron and/or othermetals such as cobalt and copper), phosphorus (e.g. phosphate) and/orsulphur (e.g. sulphate). Preferably the aqueous liquid will have a lowsulphur content, for example less than 3.0 g, preferably less than 2.0 gor 1.0 g, of sulphur by liter of aqueous liquid.

Preferably, the pH, temperature and/or oxygen content (of the aqueousliquid) during fermentation is controlled. This may be to keep the pH,temperature and/or O₂ content constant or within a desired range. Inthis respect, the fermented vessel may have pH, temperature and/or O₂content sensors.

The pH of the aqueous liquid during fermentation may be from 2 to 8,such as from 3 to 7, optimally from 4 to 6.

The temperature of the aqueous liquid during fermentation is notparticularly critical, but may be from 20 to 50° C., such as from 25 to40° C., optimally from 30 to 35° C.

It is important that during fermentation mixing occurs. In other words,the aqueous liquid and fungal cells are suitably either mixed oragitated. This may be achieved if aeration is provided, in other wordsby bubbling air into the aqueous liquid. This may serve the additionalpurpose of providing oxygen to the fungal cells: hence the fermentationis preferably an aerobic one.

Other means of agitation or mixing include stirring, for example usingan impeller. This may be of a hydrofoil axial flow design or may bedesigned so that the aqueous medium is forced radially outwards from theimpeller (such as a turbine). Even if there is no stirring it ispreferred that the fungi are provided with oxygen during fermentation,and so aeration (e.g. by bubbling air, O₂ or other oxygen-containinggas) is advantageous here. Aeration may be at from 0.1 to 2.0, such asfrom 0.5 to 1.0 vvm.

One of the advantages of aeration and/or agitation is that the oxygencontent of the aqueous liquid can be kept relatively high. This may beat least 10%, such as at least 15%, optimally at least 20% (in terms ofair saturation). This allows a more efficient formation process, and canthus result in a quicker and/or higher content of fungal cells and/orfungal protein. This is particularly advantageous for fungal cells usedin the invention because these are sufficiently robust to allowagitation and/or mixing during fermentation. This is not always possiblehowever with (the more sensitive) fungal cells of the group Fusarium, asthe art teaches the use of airlift fermentors, which do not havemechanical stirrers, with such organisms. Thus with most Mucoralesorganisms one does not have to use expensive equipment, such as airliftfermenters, developed for other (less robust) organisms, which means theedible substance can be produced more cheaply.

The fermentation may take from 1 to 12 days, such as from 2 to 6 or 7 to10 days, and optimally from 2 to 4 days. A shorter fermentation lendsitself towards a batch, rather than continuous, fermentation process.

Once fermentation has finished, or fermentation is to be stopped, watercan be removed from the combination of the fungal cells and thesurrounding liquid produced. In the art this combination of aqueousliquid and fungal cells is often referred to as a “broth”. Duringfermentation the vessel should contain only this broth, and this ispreferably entirely liquid (and so devoid of any solid material). Thecells may be rinsed, such as with an aqueous liquid e.g. water, beforeor after this water removal stage, and either or both may result in theseparation of the cells from extracellular matter (e.g. protein) ifdesired. If necessary depelleting (the dispersion or minimisation of anypellets in the liquid) may be conducted before water removal (Forexample by sonication or shear mixing).

Water removal is preferably by mechanical means or by mechanicaltechniques. These include various solid-liquid separation techniquessuch as mechanical de-watering, filtration, centrifugation (preferred),settling (in other words, the material is allowed to settle, thus usinggravity), heating or drying.

After this de-watering the water content can be from 50 to 90%, such as60 to ⁸⁷%, optimally from 75 to 85%.

Following this (optional) de-watering, the RNA content of the fungalcells can then be reduced. This can be achieved by chemical and/orphysical methods. The preferred method is to use and so take advantageof one or more enzymes already inside the fungal cells to digest theRNA. This may allow any resulting (small) RNA molecules (or degradationproducts thereof) to pass through, and so outside, the cell membrane.Suitably the (undesirable) nucleotides inside the cell are cleaved into2, 3 and 5-nucleotides; thus it may be these nucleotides that aretransported through the cell membrane. Dewatering may also remove othercompounds not desired during further processing, such as glucose (forexample due to later heat treatment).

A preferred method of RNA removal is heat treatment, in other wordsheating the fungal cells. This may have two effects. Firstly, the cellbecomes more permeable, allowing RNA and other molecules to pass outsidethe cell. It may also increase the activity of nucleases, such asRNAases, inside the fungal cells. A further advantage is that such heattreatment may inactivate any undesirable enzymes inside the fungalcells. Alternatively or in addition (ribo)nucleases and/or RNAses may beprovided or added, rather than just relying on enzymes inside the cells.

The preferred RNA reduction technique therefore involves the transfer ofRNA from inside the fungal cell to the outside of the fungal cell, forexample into a surrounding aqueous liquid (e.g. the broth). The cellscan later be separated or removed from this aqueous liquid.

If heat treatment is employed for RNA reduction, the fungal cells can beheated to a temperature of from 40 to 80° C., preferably from 50 to 70°C., optimally from 55 to 65° C. This may be for a time from 20 to 50minutes, preferably from 25 to 40 minutes, optimally from 25 to 35minutes. The temperature of the heat treatment to enable RNA reduction,and the length of time for which this temperature is maintained can beimportant. If the temperature is too low, this may not active enzymesinside the cells that will reduce RNA content. Similarly, if thetemperature is too high, then this will denature or otherwise result ininactivation or such enzymes. A balance therefore needs to be struck,and a temperature chosen between these two extremes. The conditions arethus preferably such that enzyme(s) inside the cells are activated orallowed to reduce the RNA content of those cells. It may not be enoughto simply increase the temperature from, for example, 30 to 100° C.,because in doing so the cells may not be long enough at an intermediatetemperature which allows the enzymes inside the cells to reduce the RNAcontent. Thus, depending on the organism in question, a temperature andtime is chosen so that RNA reduction occurs: although there are priordisclosures of heating fungal cells these conditions may not be suitablefor effecting RNA reduction (because either the temperature or the timeat that temperature will not allow the enzymes inside the cells toreduce the RNA content).

One other method of RNA removal is to subject the fungal cells to anacid or alkaline pH. If an acid pH is provided, this may be from 3 to4.5, such as from 3.5 to 4.2. This acid treatment may last from 15 to120 minutes, such as 30 to 60 minutes. It may, if necessary, be combinedwith heat treatment, such as from 40 to 60° C., such as from 50 to 60°C. Suitable acids include inorganic acids such as hydrochloric acid,phosphoric acid, nitric acid and/or sulphuric acid. A fungicide may alsobe used to kill the cells instead of or in addition to the methodsmentioned.

If an alkaline pH is provided, this may be from a pH of 8 to 12, such asfrom 9 to 11, optimally at a pH of from 8 to 10. The alkali may beprovided by ammonia, alkali or alkaline earth metal oxides, hydroxidesor carbonates. The alkali treatment may be for the same time asspecified for the acid treatment, and may optionally also be accompaniedby heat treatment as described for the acid treatment. However in somecases a lower elevated temperature may be more appropriate, for examplefrom 40 to 80° C., such as from 60 to 70° C., optimally from 62 to 68°C.

The fungal cells may be subjected to both acid and alkali treatment,either of which may be combined with heat treatment. Preferably thoughthe RNA reduction is a one stage process. The acid or alkali solutionused to contact the fungal cells may be discarded, reused or recycled.

If necessary the RNA removal may be followed by pasteurization or a heatshock treatment. This may involve particularly high temperatures, suchas 100 to 150° C. or 40 to 120° C., optimally 130 to 140° C. or 40 to60° C. This may only last for 30 to 200 seconds, such as from 80 to 120seconds. For the higher temperature ranges: it may be from 5 to 120,e.g. 20 to 50 minutes for the lower temperatures. This heat shocktreatment may be provided after acid and/or alkali treatment.

Thus heat treatment and/or pasteurization may be in addition to heatingfor RNA removal. This may be necessary if the RNA removal does not killall the cells. Alternatively this stage could be thought of assterilization or the inactivation of undesirable proteins or enzymes,for example proteases, lipases, amylase, phospholipases and/orlipoxygenases. This step (as with RNA reduction) may be performed eitherif the fungal cells are still in an aqueous liquid (for example thebroth, e.g. while still in the fermented vessel) or (preferably) if theyhave been subjected to one or more water removal steps. Here the heattreatment may either reduce water content and/or make the fungal cellsmore water-insoluble.

Whichever RNA content reduction technique(s) are employed, it isdesirable that the fungal cells remain intact, or whole, in other wordsare not lysed. The cells should thus be intact but not alive (e.g.killed or non-viable).

The fungal cells after RNA reduction preferably have an RNA contentbelow 4.0% or 2.0%, such as from 0.1 to 2.0%, preferably from 0.5 to1.5%. Optimally the RNA content is from 0.4 to 0.8%. These percentagesare based on the dry weight of the cells. Cells with a reduced RNAcontent may thus have a content below that of the naturally occurringfungus or the fungus used in the fermentation process (stage (a) of thefirst aspect).

The protein produced may be located in various parts of the fungal cell.It may represent up to 30%, such as up to 40% and optimally up to 50% ofthe fungal cell itself (based on dry weight). The fungal protein may beinside the cell (intracellular) or inside the cell wall. The former mayinclude two different “types” of proteins, for example structuralproteins (those concerned with DNA; ribosomes; membranes etc) andcatalytic proteins (for example enzymes). Cell wall proteins include notonly those that are inside or part of the cell wall, but may be outsideof the cell wall but still bound to the cell wall. This is contrast tosecreted (e.g. extra cellular) proteins that are not bound to the cell,and which are usually discarded or otherwise lost during processing. Thematerial containing the fungal cells may then be subjected, ifnecessary, to a (further) water removal step, or de-watering. This willpreferably reduce the water content to from 50 to 90%, such as from 60to 85%, optimally from 75 to 85%. This may be after one or more rinsingor washing steps (for example with water, e.g. tap water). The resultingmaterial may have a dry matter content of from 10 to 40%, preferablyfrom 15 to 35%, optimally from 20 to 30%.

The liquid removed at this stage preferably contains the RNA, RNAdegradation products or any other undesired substances either removedfrom the cells or transferred from the inside the outside of the cells,in the previous RNA reduction or heating stage(s). Procedures forremoving the water here are the same as described for the optionalremoval step earlier following fermentation. However, at this stagefiltration is preferred, such as vacuum filtration.

At this stage in the process one can have produced the proteinaceoussubstance that is the subject of the third aspect of the invention. Itmay be in the form of an (e.g. aqueous) paste, a biomass or a filtercake. Further processing, in particular texturising, for example usingmechanical methods, can then be performed in order to produce the edible(proteinaceous) textured product of the fourth aspect. Other processingtechniques may include chemical, physical and/or enzymatic treatment.

To the substance of the second aspect one may add or mix in one or moreedible component(s). These may be to add texture and/or flavour.Preferred components include hydrocolloids, for example pectin, starch,carrageenan or alginate. This may be before or after mechanicalprocessing step(s) such as milling, crumbling, cutting, kneading and/orhomogenising.

Also contemplated are proteins, for example milk protein such as casein,ovoprotein such as egg albumin or eggs themselves (yolk and/or eggwhite), vegetable proteins such as soy, or cereal proteins, such asgluten, or enzymes (e.g. proteases, phosphodiesterases).

Other edible components include flavour enhancers such as salt, sugar,IMP and/or GMP (although in this case it will be preferred that the RNAlevel does not exceed those mentioned earlier for the fungal cells),flavouring agents such as spices, herbs, proteins (e.g. from 2 to 5%such as a milk protein, e.g. casein, a vegetable protein, an ovoprotein,e.g. albumin), hydrocolloids (e.g. from 5 to 20% such as pectin,carageenan, agar, xanthan, gellan, galacturonic or mannuronic acid orsalts thereof), flour, alginate (such as 0.2 to 1.0%), edible polymers(e.g. cellulose, methylcellulose), gelling agents (such as egg albumin,whey protein and alginate), polysaccharides (such as from 0 to 10%, forexample starch or pectin), colouring agents, plant material such asvegetables (onions, carrots, soy, peas, beans or cereals such as wheat,oats, barley) and emulsifiers. It may also include meat-likeflavourings, such as beef, pork or poultry (chicken or turkey)flavourings or other non-meat products. Additional components may beprovided to improve taste (organoleptic properties) to improve waterbinding, fat binding, emulsification properties, texture, volume,viscosity, flavour, aroma and/or colour (dyes, carotenoids, etc.). Eggalbumin may be included to improve whippability, colouring or as abinder of other proteins. Egg yolk can be used for emulsification,colour or flavour. Soy protein can be employed for water binding, fatbinding and to improve texture. Gelatin can be included to improvegelation. Milk protein or salts thereof for water binding and fatbinding flavour or texture and wheat gluten for water binding, textureor flavour. The edible proteinaceous product may therefore be used toreplace or be provided in addition to one or more of the following:vegetable proteins, egg white, gelatin, edible proteinaceous foamingagents and milk proteins. Fibrous materials may also be included (e.g.vegetables such as onions).

The texturization is intended to provide texture to the product so thatit has meat-like texture and/or it has a mouthfeel similar to meat. Itmay thus have a fibrous or meat-like appearance.

Texturization is preferably by one or more mechanical means. Theseinclude milling, crumbling, mincing, slicing, cutting (e.g. into chunks,slices or layers), kneading, layering, rolling, sheeting and/orextruding. Preferably it may result in the alignment of the fungalprotein into fibres, which may assist to give the product the appearanceof meat.

The texturising may however comprise physical methods, for exampleheating and/or freezing. Both of these techniques may also result infurther water removal. Freezing in particular may assist in alignment ofthe fungal cells into a fibrous appearance.

The mechanical shaping may include placing the mixture of fungal cellsand edible component(s) into a mould or other container of a desiredshape, and then cooling (such as freezing) and/or heating (for example70 to 100° C., to cause gelling, for example) by various methods such assteaming, boiling and/or frying. Pressure may be applied if necessary.The material can then be removed from the mould or container, and canretain the shape of that container.

The shaped product may for example be in the shape of animals, birds orfish, letters of the alphabet, numbers, etc which may be particularlysuitable for foodstuffs for children.

The textured product may also be in the form of a dried powder, whichmay be included in a foodstuff to increase mouthfeel or to increaseviscosity.

A particularly preferred texturization method involves granulation, forexample to produce granular particles. Before any texturization, thecombined fungal cells and fungal protein may have an average watercontent of from 15 to 85%. After texturing (e.g. granulation), theresulting granules may have an average water content of below 30%, e.g.less than 20%, optionally less than 10%.

Preferably granulation is achieved using extrusion. This is preferredbecause extrusion conditions can be adjusted to minimise disruption ofthe fungal cells. Extrusion may therefore be conducted without heating,for example at from 15 to 85° C. During extrusion the granules may formnaturally, falling away under their own weight (from the die plate, suchas by gravity) or one can use a cutter, such as a rotating blade, to cutthe long strands of “spaghetti” produced by the extrusion. Followingextrusion the granules preferably have a water content less than 15%,such as less than 10%, and optimally from 3 to 7%. The granules may havea diameter of from 0.3 to 10 mm, such as from 0.7 to 5 mm, optimallyfrom 1 to 3 mm.

Extrusion may thus be used to form elongate “spaghetti” like products(these may be cylindrical and/or of circular cross-section) if passedthrough a suitable die-plate (e,g, with circular or square holes).However formation into for example sheets or layers can be achieved bypassage (e.g. using extrusion) through one or more slots. These formsmay also be prepared by the use of one or more moving surfaces, such asroller(s) and/or cylinder(s). These may be moving in the same directionor counter-rotating and there may be one, two or up to five suchsurfaces.

The proteinaceous product may therefore be in a variety of forms. Forexample, it may be in the form of chunks, for example meat-like chunks,dough, sheets, granules, extrudate, slices or may be layered. Theseforms may be dried or frozen. The product may be included into thefoodstuff with no or additional processing. They may be recognizable aschunks in the foodstuff, and may have the appearance of meat.

They can thus be included in foodstuffs as meat substitutes, andfoodstuffs contemplated include pies, microwaveable meals, savourysnacks, sausages, patties, burgers, spreads and pâté, dried powder (e.g.for soups).

The textured product may be in the form of pellets or granules, andthese too may be dried or frozen. They may be adapted for dehydrationbefore consumption. These products may be included in soups or sauces.The product (e.g. pellets or granules) may be included in burgers orsausages for example with an (e.g. edible) binder. A suitable sausagepreparation process and sausage making machine is described in theInternational patent application no. PCT/EP99/02795 filed on 26^(th)Apr. 1999 in the name of Gist-brocades B. V.

A particularly preferred process of the present invention may thereforecomprise:

-   -   1. fermenting fungal cells of the order Mucorales, for example        in an aqueous liquid contained in a fermenter vessel, the liquid        comprising assimilable nitrogen and carbon sources. The liquid        and cells can be mixed and/or aerated during fermentation and if        necessary depelleting can be performed;    -   2. optionally removing water, for example removing the, or water        from, the aqueous liquid, preferably using mechanical techniques        such as filtration, centrifugation (e.g. once or twice),        settling and/or drying;    -   3. reducing the RNA content of fungal cells, for example by        physical, chemical and/or enzymatic treatment(s), but preferably        by heat treatment (e.g. 60 to 75° C. for 25 to 35 minutes;    -   4. heat treating, pasteurizing or killing the cells or otherwise        (e.g. chemically) inactivating undesirable proteins or enzymes        inside the fungal cells;    -   5. optionally, removing water (e.g. if not done in stage 2),        such as to provide the edible (e.g. proteinaceous) substance;    -   6. adding to the fungal cells (or edible substance) one or more        edible components;    -   7. texturising the fungal cells (either before (e.g. milling or        crumbling) or after (e.g. kneading or extruding) edible        component addition in stage 6), for example using mechanical        processing;    -   8. subjecting the fungal cells to physical treatments such as        heating (e.g. boiling, steaming, frying) and/or freezing, or        otherwise removing water;    -   9. optionally before or after stage 8, shaping and/or otherwise        mechanically processing (if necessary) to give a textured        product; and    -   10. including or processing the edible product into a foodstuff        or supplementing a foodstuff with the product.

The foodstuff can comprise a textured edible product either of thefourth aspect or preparable by a process of the third aspect. As will beexpected, the foodstuff may contain one or more additional ediblecomponents or ingredients in addition to the fungal cells. These may bethe same as those described above in relation to the proteinaceousproduct.

The textured product can be included into the foodstuff as it is, inother words it may simply be used to supplement an existing foodstuff orit may be used in the preparation of a foodstuff. It may be heated firstto generate nicer flavours or to brown the product.

Preferred foodstuffs include ready-made or convenience meals, ormicrowavable meals, burgers, pies, pasties, sausages and soups. Theproduct can be used a substitute for meats such as pork, beef, poultry,game, ham, veal or even fish.

These foodstuffs are of course intended for human consumption, althoughfoodstuffs for animals, in particular pets (such as dogs and cats), suchas canned foodstuffs, or farm animals (pigs, cows, sheep etc) arecontemplated.

Other foods can be included as components or ingredients, for examplerice and pasta.

Preferred features and characteristics of one aspect of the inventionare applicable for another aspect mutatis mutandis.

The invention will now be described by way of example with reference tothe accompanying Examples, which are provided merely for the purposes ofillustration and are not to be construed as being limiting.

EXAMPLES Comparative Example 1 Selection of Suitable Microorganisms

The microorganism needs to be food grade and the substance shouldcontain “valuable” proteins. The essential nutrients as in meat shouldalso preferably be present. The morphology/structure of the biomass hasto be suitable to produce a mycoprotein enriched product with a “bite”and organoleptic sensation of meat-like products.

The Examples demonstrate that manufacturing fungal food from Mucoralesfungi is feasible, and that the more “primitive” families within theMucorales order can be preferable.

Advantages of Mucorales fungi include:

-   -   1. low or absent mycotoxin production;    -   2. simple and cheap biomass production: good growth to high        concentrations in clear media (composed of salts, a well-defined        complex N-source, and glucose or oligosaccharides);    -   3. down-stream processing procedures are acceptable for        foodstuffs; and    -   4. good quality of end product.        Flask Experiments

In flask experiments various strains were tested belonging to theMucorales families of Choanephoraceae, Mucoraceae, and Mortierellaceaeto test their growth in simple and clear media.

Growth was tested in several different media including the twosemi-defined media:

compound concentration yeast extract or peptone 5 g/kg glucose 30 g/kgpotassium phosphate 0.10 M ammonium sulphate 0.1 M magnesium sulphate1.25 mM zinc sulphate 0.03 mM manganese sulphate 0.2 mM iron chloride0.09 mM copper sulphate 0.03 mM

The components were dissolved in deionized water, and sterilized for 20minutes at 120° C.: the glucose was sterilized separately. The pH aftersterilization was 6.0.

The experiments were conducted in Erlenmeyer flasks (100/500 ml).Inoculation took place with a suspension of spores prepared freshly bygrowing the strains for several days on a malt agar surface, rinsing thespores from the surface and storing them in a freezer. The flasks wereincubated between 25 and 35° C. for 2 to 4 days on an orbital shaker(with a 2.5 cm stroke at 250 rpm).

The following strains were tested:

species family source Blakeslea trispora Choanephoraceae CBS 130.59Gilbertella persicaria Choanephoraceae CBS 247.59 Absidiapseudocylindrospora Mucoraceae CBS 100.2 Phycomyces blakesleeanusMucoraceae CBS 226.92, NRRL 1555 Rhizopus oryzae* Mucoraceae ownisolate* Mucor hiemalis Mucoraceae CBS 242.35 Rhizomucor miehei*Mucoraceae own isolate* Mucor rouxii Mucoraceae CBS 416.77 Mortierellaalpina* Mortierellaceae own isolate* *Strains are commerciallyavailable, e.g. from the CBS (Centraal Bureau voor Schimmelcultures,Delft, The Netherlands).

All strains grew well within several days, usually in a mixed form ofboth filamentous mycelium and pellets. In all cases it was possible toobtain at least 5 g biomass/liter of broth in the course of incubation,measured by filtering the biomass and weighing it after drying for 24hours at 105° C. on a preweighed filter paper. The presence of pelletswas checked visually.

Comparative Example 2 Fermentor Experiments

As part of the scale-up process all the strains from Example 1 weresubjected to lab scale fermentor experiments. The objective was to testthem for growth in simple media, growth to high biomass concentrationthat allows further scale-ups and growth in a form that allows inclusionin a foodstuff.

The experimental set up was as follows, starting with inoculumpreparation.

The spore suspension was prepared as described in the previous Example.With this spore suspension an inoculum culture was started, using a soybean meal based medium to promote hyphal growth (soy flour 15 g/kg,yeast extract 5 g/kg, K₂HPO₄ 1 g/kg and glucose. H₂O 20 g/kg). Themedium was sterilized for 45 minutes at 120° C. in Erlenmeyer flasks atpH6. As soon as full growth had been reached the culture was transferredto a lab fermentor, containing medium that was prepared using thefollowing components:

component concentration (g/kg) yeast extract 1 glucose 20 ammoniumsulphate 6 magnesium sulphate.7 H₂O 2 calcium chloride 0.5 potassiummonophosphate 3 zinc sulphate.7H₂O 0.0144 iron sulphate.7H₂O 0.15manganese sulphate.1H₂O 0.0228 copper sulphate.5H₂O 0.0024 cobaltsulphate.7H₂O 0.0038 thiamine.HCl 0.004 nicotinic acid 0.002

All compounds were dissolved in deionized water and mixed, except theglucose and the phosphate which were prepared separately. The pH wasadjusted to 6.0 or 4.5 using NaOH, and the medium was sterilized in thefermentor for 45 minutes at 121° C. in an autoclave. The glucosesolution and phosphate solution were added after separate sterilizationfor 20 minutes at 120° C., the first after acidification to pH 5 withphosphoric acid.

Next to the batch medium a carbohydrate feed was supplied whichconsisted of glucose at a concentration of ca. 500 g/kg. The preparationwas as described for the glucose solution of the batch medium.

The fermentor was equipped with temperature, pH and foam controls. Toadjust the pH, solutions of ammonia and sulphuric acid were used.Dissolved oxygen concentration and the composition of the liberated gaswere measured. The culture was aerated using ca. 1 volume of air pervolume of broth per minute. Mixing was intensive using Rushton turbinesand baffles. The glucose feed was applied at a rate between 1 and 5 g ofglucose/kg broth/hour and started when the glucose concentration in thebroth had decreased to a concentration below 5 g/kg.

Samples were taken twice every 24 hours for off-line analysis ofconcentrations of unused substrate, biomass and by-products. Microscopicinspection was also performed.

The following strains, thus tested in flasks for experiments in labscale fermentors, were selected:

Rhizopus oryzae, Morrierella alpina, Blakeslea trispora, Gilbertellapersicaria and Absidia pseudocylindrospora.

In all cases the biomass accumulated to concentrations from 20 to 50g/kg within 80 hours of cultivation.

All strains thus indicated potential to be able to produce biomass in asimple medium and at a low cost when scaling up the process.

Comparative Example 3 Morphology Analysis

In shake flasks various microorganisms were cultivated according to theprocedures given in Example 1. The morphology of the biomass wasexamined by light microscopic methods. The characteristics found areshown in Table 1. The morphology of the Mucorales organisms wasdifferent to that of the Fusarium species.

TABLE 1 Strain Branched Fusarium graminearum (now reclassified as F.venenatum, no IMI 145425) Mortierella alpina yes Gilbertella persicariayes Rhizopus oryzae yes Absidia pseudocylindrospora yes

Examples 4 and 5 and Comparative Example 6 Lab Scale Fermentation andBiomass Analysis

In Example 4, lab scale fermentors were used to cultivate threemicroorganisms (Absidia pseudocylindrospora, Gilbertella persicaria andMortierella alpina) according to the procedures described under Example2 and the same conditions were used to culture Fusarium venenatum(Comparative Example 6).

In Example 5 Rhizopus oryzae was cultured on a production scale (fordetails see Example 8: a part of the broth was used for the followinganalysis).

The following recovery procedure was used to first prepare a biomassfiltercake:

-   -   centrifugation and washing the biomass (to remove excess medium        components like glucose);    -   heat treatment at 65° C. to reduce enzymatic activity and to        reduce RNA;    -   heat treatment to 90° C. to pasteurise the broth;    -   filtration on a lab filter press, including washing with tap        water; and    -   packing and storage.

Centrifugation. Portions of 1 liter of biomass were centrifuged in aBeckmann centrifuge (type J-6M/E) for 5 minutes at 5000 rpm. Thesupernatant was decanted and discarded. The pellet was resuspended intap water and recentrifuged. The supernatant was decanted again. Thewashed pellet was resuspended in tap water.

Heat treatment (RNA reduction): the broth was heated to 65° C. and keptat this temperature for 30 minutes.

Heat treatment (to kill enzymes): the broth was further heated to 90° C.and kept at this temperature for 30 minutes.

Filtration: the heated broth was filtered in a 2 liter filter press(type Seitz Enzinger Noll, Germany) provided with a polypropylene filtercloth at a starting pressure of 0.5 bar.

The resulting cake was washed with 10 cake volumes of tap water. Afterwashing the cake was blown dry with air at 2 bar for 15 minutes. Thecake was collected for further treatment. The cake was analysed for drymatter, RNA, crude protein (Kjeldal-N) and fat. The data resulting fromanalysis is given in Tables 2 and 3.

TABLE 2 start total dry matter volume cake of cake Protein fat RNAExample Strain (ml) (g) (%) (N × 6.25) % (%) (g/kg) 4 Mortierella alpina3250 282 26 13.0 4.46 2.85 4 Gilbertella persicaria 3700 1135 15.3 5.32.54 0.77 4 Absidia pseudocylindrospora 3400 284 20.6 9.9 3.44 4.45 4Gilbertella persicaria 2940 349 16.5 7.2 2.07 3.68 5 Rhizopus oryzae10000 682 16.1 6.9 1.95 4.82 6 Fusarium graminearum 3500 552 24.6 16.12.12 13.1 (Comp)

TABLE 3 (Calculated on dry matter) protein fat RNA Example Strain (%w/w) (% w/w) (% w/w) 4 Mortierella alpina 50.0 17.1 1.1 4 Gilbertellapersicaria 34.6 16.6 0.50 4 Absidia 48.1 16.7 2.1 pseudocylindrospora 4Gilbertella persicaria 43.6 12.5 2.2 5 Rhizopus oryzae 43.3 12.2 0.3 6Fusarium graminearum 65.5 8.6 5.3 (Comp)

Example 7 Pilot Plant Production

Fermentations of the Rhizopus oryzae strain used in Example 2 was scaledup in a pilot plant fermenter with a working volume of 3 m³ from theconditions as described in Example 4. After fermentation the broth wascooled to 5–10° C. and harvested.

A part of the broth (100 liters) was centrifuged in a Westfalia NA7 discseparator after dilution with tap water to 500 liters. The centrifugewas provided with 4 nozzles, each of a diameter of 1 mm. Two streams offluid were obtained. The supernatant (400 liters) was discarded and aconcentrate stream that contained the biomass (fungal cells) retained.The concentrate was diluted to the original volume with a 100 mMsolution of K₂HPO₄.

The mixture was recentrifuged and the supernatant was discarded.

The washed concentrated biomass was then heated to 65° C. for 30minutes. The concentrated biomass was further heated to 90–95° C. andkept at that temperature for 30 minutes.

Part of the resulting broth (1.35 m³) was filtered in a Schule membranefilter press with a filtration area of 6 m² and a pressure of 0.3–2 bar.The obtained filter cake was washed with cold tap water (5–10° C.). Thefilter cake was squeezed by the membranes at a pressure of 6 bar. Thisresulted in 73 kg filter cake with a dry matter of 24.8%.

Example 8A Preparation of Edible Biomass

Three production fermentations the Rhizopus oryzae strain used inExample 2 were performed in a standard production fermenter with aworking volume of 30 m³. The fermenter had a pH control, a Rushtonturbine with adjustable speed, air supply, foam control and temperaturecontrol. At harvest the production microorganisms were killed and theRNA content reduced by heating the biomass to 50–55° C. with directsteam in the presence of 1 g/l benzoic acid at pH 4.5–5.0. Afterreaching 50–55° C. the broth was cooled to below 20° C. The broth wastransferred to another vessel, diluted with cold tap water and furthercooled to 4–6° C. The cooled broth was then filtered in a Schenkmembrane filter press with a working cake volume of 2.5 m³ and the cakewashed with 20 m³ of cold tap water. The cake was squeezed by applyingpressurized water in a membrane system (4–9 bar). The cake wasdischarged from the filter press and part of this crumbled, packed inbags and frozen in a cold store at −15 to −20° C. Part of the frozenbiomass was cut into particles having a size of from 1–3 mm and two orthree samples from each of the three fermentations were taken for thefollowing analysis (Table 4).

TABLE 4 Squeeze Fermentation Pressure Total Cake % dry matter No. SampleNo. (bar) (kg) (w/w) 1 1 9 362 50–55 1 2 9 383 50–55 2 1 4 385 45–50 2 24 404 42–46 2 3 4 437 42–46 3 1 4 449 42–46 3 2 4 392 36–40

The samples were further analysed chemically (for RNA, crude protein (asKjeldahl-N), fat, mycotoxins and other components) and gave thefollowing results (Table 5).

TABLE 5 Fermentation No. - Sample No. 1-1 1-2 2-2 2-3 3-1 Dry matter (%)52.1 52.1 42.5 42.9 41.1 Ash (%) 1.6 1.6 1.3 1.1 1.8 Total crude fibres(%) 30.1 30.1 27.4 27.5 27.0 Protein (as N × 6.25) (%) 43.2 43.2 39.740.1 43.3 Total fat (%) 12.7 12.7 16.8 16.8 12.2 RNA (mg/kg) 4222 nd1132 nd 2518 RNA (%) 0.4 nd 0.1 nd 0.3 Carbohydrates 12.5 12.5 14.9 14.515.8 Total amino acids % 26.11 nd 23.90 nd 25.13 calculated Total fattyacids % 11.9 nd 15.7 nd 11.5 calculated Mycotoxin (μg/kg) Aflatoxine B1<2.0 <2.1 <2.3 <2.3 <2.4 Aflatoxine B2 <2.0 <2.1 <2.3 <2.3 <2.4Aflatoxine G1 <2.0 <2.1 <2.3 <2.3 <2.4 Aflatoxine G2 <2.0 <2.1 <2.3 <2.3<2.4 Ochratoxin A <2.0 <2.1 <2.3 <2.3 <2.4 T2 Toxin <200 <210 <230 <230<240 Zearaleone <20 <21 <23 <23 <24 nd = not doneAmino acid composition.

TABLE 6 Fermentation No. - Sample No. Amino acid (%) 1-1 2-2 3-1Methionine 0.56 0.52 0.54 Lysine 1.92 2.06 2.07 Cysteine 0.44 0.35 0.36Asparagine (acid) 2.99 2.55 2.51 Threonine 1.52 1.39 1.44 Leucine 2.152.10 2.17 Isoleucine 1.38 1.42 1.44 Serine 1.50 1.25 1.31 Glutamine 3.202.69 2.87 Glycine 1.25 1.13 1.24 Alanine 1.55 1.49 1.65 Valine 1.67 1.751.70 Tyrosine 1.13 1.06 1.12 Phenylalanine 1.34 1.23 1.36 Histidine 0.670.61 0.68 Arginine 1.36 1.04 1.14 Praline 1.04 0.83 0.97 Tryptophan 0.440.43 0.46

Example 8B Dried Biomass

The remaining cut particulate biomass for Example 8A was dried inportions or 30–50 kg in a Aeromatic T4 fluid bed dryer with a bottomplate area of 0.26 m² by means of dry air of a temperature of from55–65° C. Drying terminated at a bed temperature of 38–40° C. Samples ofthe dried biomass had the following dry matter contents (Table 8).

TABLE 7 Fermentation No. Sample No. dry matter content (%) 2 2 93.2 3 195.5

Examples 9A, 9B and 9C Sheeting, Layering, Rolling

The filter cake from Example 8A was milled and crumbled in portions ofapproximately 25 kg by a Lödige high shear mixer for 5 minutes. To thecrumbled cake 1 kg of egg albumin (Example 9A) was added and the mixturekneaded. The procedure was repeated with a little water and spices beingfirst mixed with the egg albumin (Example 9B).

The mixture was formed into sheets of 1 mm by rolling equipment.

The sheets were heated to 80° C. in an ventilated oven or tunnel. Thesheets were layered and rolled in the form of a “Swiss roll” and theroll frozen to −20° C. using liquid carbon dioxide.

The same procedure was repeated except 1 kg pectin (Example 9C) wassubstituted for the egg albumin.

Examples 10A to D Burgers

To the biomass from Examples SA and 8B colouring additives, tasteenhancing products (spices, vegetables and onions) were added. Themixture was then homogenised in a kneader and the homogenised mixtureformed into burgers, pasteurised and packed. Both procedures wererepeated with egg albumin being first mixed with the taste enhancers.

Examples 11A to D Sausages

To the biomass from Examples SA and B colour additives, spices,vegetables (onions) were added. The mixture was homogenised in akneader. The homogenised mixture was extruded into a continuous tube (sothat it formed the interior of the sausages) while co-extruding a(vegetarian) skin-forming material using a continuous sausage-makingsystem (Stork) to make sausages. The two procedures were repeated withegg albumin being first mixed with the colour additives and spices.

Examples 12A and B Granules

The filter cake from Example 8A was milled and crumbled into portions ofapproximately 25 kg by a Lödige high shear mixer for 5 minutes. To thecrumbled cake 1 kg of egg albumin was added and the mixture kneaded. Thekneaded mixture was extruded with a single screw extruder with adieplate with holes of 1 mm. The extrudate was transported by a belt anddried in a fluidised bed drier (air temperature of 50° C.) to formgranules. For Example 12B pectin was used in the same amount instead ofegg albumin.

Examples 13A to D Burgers

A batch of 25 kg of the dried extrudate from each of Examples 12A and Bwas mixed with 60 kg tap water. To this mixture the food additives usedin Example 10 (both with and without egg albumin) were added and themixture kneaded and formed into burgers, pasteurised, packed and frozen.

Examples 14A to D Sausages

A batch of 25 kg of the dried extrudate from each of Examples 12A and12B was mixed with 60 kg tap water. To the mixture the food additivesfrom Example 11 (both with and without egg albumin) were added and themixture processed into sausages as described in Example 11.

Examples 15A and B Burgers

To biomass 25 kg from each of Examples 8A and B was added colouradditives and taste enhancing products (spices, vegetables, onions). Tothe mixture 1 kg vegetable fibres (cellulose fibres with an averagefibre length of 300–1000 μm) was added and homogenised in a kneader. Thehomogenised mixture was formed into burgers, packed, pasteurised andfrozen.

Examples 16A and B Sausages

To biomass (25 kg) from each of Examples 8A and B colour additives,spices, vegetables and onions were added. To the resulting mixture 1 kgvegetable fibres (cellulose fibres with an average fibre length of300–1000 μm) was added and the mixture homogenised in a kneader. Thehomogenised mixture was formed by extrusion into sausages byco-extrusion with a vegetarian skin as described in Example 11.

Examples 17A and B Granules for soups

The filter cake from Example 8A and B was milled and crumbled inportions of approximately 25 kg by a Lödige high shear mixer for 5minutes. To the crumbled cake a mixture of 1 kg egg albumin was addedand 1 kg vegetable fibres (cellulose fibres with an average fibre lengthof 300–1000 μm). The mixture was kneaded and then extruded in a singlescrew extruder with a dieplate with holes of 1 mm. The extrudate wastransported by a belt and dried in a fluidised bed dryer (airtemperature of 65 to 80° C.), to form granules. These were then added toa soup and dried to form soup powder (that produces soup onrehydration).

Examples 18A and B Burgers

A batch of 25 kg of the dried extrudate from Examples 17A and B wasmixed with 60 kg tap water. To the mixture the food additives describedin Example 15 were added and the mixture kneaded and used to makeburgers as described in Example 15.

Examples 19A and B Sausages

A batch of 25 kg of the dried extrudate from Examples 17A and B wasmixed with 60 kg tap water. To the mixture the ingredients as describedin Example 16 were added, the mire kneaded and used to form sausages asdescribed in Example 16.

Examples 20 to 35 and Comparative Examples 36 to 38 Patties, Sausagesand Mini-Burgers

A dough was prepared by mixing and cutting the biomass prepared inExamples 4 and 5 in a lab scale food processor (Braun Combi type 700).Water and various edible ingredients (amounts given below) were addedand mixed into the biomass in the food processor. The dough was placedin moulds (patties or burgers) or used to fill casings (sausages).

The shaped doughs were heated to 80° C. (internal dough temperature)either by steaming (patties), boiling in a water bath (sausages) orfrying (mini burgers). The products were chilled to 4–7° C. for 2 hoursand then kept for 1 week in a freezer at −20° C.

The following dough formulations were prepared (figures are in grammes).

TABLE 8 Ingredient Patties Mini-burger Sausage biomass (25% dry weight,75% water)* 53 53 53 water* 35 35 35 whey protein 2.0 2.0 egg albumin 62.0 2.0 potato starch 1.0 0.5 malt extract 0.5 0.2 dextrose 0.4 0.5 beefflavour 1.0 0.5 pork flavour 0.5 flavours (mixture of black pepper, 0.5nutmeg, coriander and garlic powder) soy oil 4.55 4.55 5.3 pectin 0.55chicken flavour 1 *NOTE: The amount of water in the biomass obtainedfrom different fermentations varied so the ratio of dry matter: waterwas adjusted so that in all cases from 13–14 g dry biomass and from39–40 g water was present.

In some cases extra pectin was added:

-   -   Patties: Mortierella, 1.5 g per 101 g dough;    -   Sausages: Mortierella, 1.5 g per 101 g dough; and    -   Burgers: Mortierella, 2.5 g per 102 g dough.

Various physical properties were noted and are shown in Tables 9 to 11.The Rhizopus oryzae foodstuffs were used as a baseline (hence the valuesare zero) and the other foodstuffs graded by comparison (+means more,−means less). For granularity, +means more granulous (i.e. lessfibrous).

Patties: Examples 20 to 25

TABLE 9 structure: biomass patty colour dough granularity juicinessfirmness Rhizopus cream/light firm 0 0 0 oryzae brown Mortierella lightbrown wet +++ ++ −− alpina Absidia dark grey wet +++ ++ −− Pseudocylin-drospora Gilbertella light brown firm ++ + + persicaria Gilbertellalight brown firm ++ + − persicariaSausages: Examples 26 to 30

TABLE 10 Sausage sausage structure: biomass colour dough granularityjuiciness firmness Rhizopus cream/light firm 0 0 0 oryzae brownMortierella light brown wet +++ ++ −−− alpina Absidia dark grey wet +++++ −− pseudocylin- drospora Gilbertella light brown firm + + +persicaria Gilbertella light brown firm + + + persicariaMini-burgers: Examples 31 to 35

TABLE 11 colour of the structure: biomass mini-burger dough granularityjuiciness firmness Rhisopus cream/light firm 0 0 0 oryzae brownMortierella light brown wet +++ ++ −− alpina Absidia dark grey wet +++++ −− pseudocylin- drospora Gilbertella light brown firm ++ + +persicaria Gilbertella light brown firm ++ + − persicaria

As is apparent different foodstuffs with varying textures can beprepared using different organisms from the Mucorales group. Forcomparison a patty, sausage and mini-burger were prepared (Examples 36to 38) using the same recipe above but using Fusarium graminearumbiomass. All three products were black in colour.

1. A process for the preparation of an edible proteinaceous substance,suitable for use in a foodstuff, comprising fungal cells, the processcomprising: a. fermenting fungal cells of the order Mucorales in anaqueous liquid contained in a fermenter vessel, the liquid comprising anassimilable nitrogen (N) source and an assimilable carbon (C) source,and mixing the liquid and cells during fermentation; b. reducing the RNAcontent of the fungal cells to below 4% by weight (wt %); c. before orafter (b), removing at least some of the water from the mixture offungal cells and aqueous liquid; and d. processing the fungal cells intoan edible substance.
 2. A process according to claim 1, wherein theliquid and fermenter vessel are devoid of an insoluble substrate for thecells.
 3. A process according to claim 1, wherein the fungal cellsconstitute at least 60% of the proteinaceous substance on a dry matterbasis or the fungal cells constitute at least 70% of the proteinaceoussubstance on a dry matter basis.
 4. A process according to claim 1,wherein the fungal cells are of the genus Rhizopus or Gilbertella.
 5. Anedible proteinaceous substance, suitable for use in a foodstuff,comprising fungal cells of the order Mucorales having an RNA content ofbelow 4 wt %.
 6. An edible proteinaceous substance, suitable for use ina foodstuff, comprising fungal cells of the order Mucorales having anRNA content of below 4 wt % which is produced by a process according toclaim
 1. 7. An edible substance according to claim 5 which is a biomass.8. An edible substance according to claim 5 which is filter cake.
 9. Anedible substance according to claim 7, wherein the biomass is milled.10. An edible substance according to claim 7, wherein the biomass iscrumbled.
 11. An edible substance according to claim 8, wherein thefilter cake is milled.
 12. An edible substance according to claim 8,wherein the filter cake is crumbled.
 13. A process for the preparationof an edible textured product, the process comprising mixing one or moreedible component(s) with an edible proteinaceous substance comprisingfungal cells of the order Mucorales having a RNA content of below 4 wt %and mechanically texturizing the mixture.
 14. A process for thepreparation of an edible textured product, the process comprising mixingone or more edible component(s) with an edible proteinaceous substancecomprising fungal cells of the order Mucorales having an RNA content ofbelow 4 wt % and texturizing to form a product of which at least 5% isfungal cells on a dry matter weight basis.
 15. A process for thepreparation of an edible textured product, the process comprising mixingone or more edible components(s) with an edible proteinaceous substancecomprising fungal cells of the order Mucorales having a RNA content ofbelow 4 wt % and mechanically texturizing the mixture, wherein theproteinaceous substance is prepared by a process according to claim 1.16. A process for the preparation of an edible textured product, theprocess comprising mixing one or more edible component(s) with an edibleproteinaceous substance comprising fungal cells of the order Mucoralesand texturizing to form product of which at least 5% is fungal cells ona dry matter weight basis, wherein the proteinaceous substance isprepared by a process according to claim
 1. 17. An edible texturedproduct, suitable for use in a foodstuff of which at least 40% is fungalcells having an RNA content of below 4 wt % of the order Mucorales on adry matter weight basis.
 18. A product according to claim 17 whichcomprises pellets, granules, a sheet, or is an extrudate, dough, roll,paste or meat-like chunk.
 19. An edible textured product, suitable foruse in a foodstuff, of which at least 40% is fungal cells of the orderMucorales on a dry matter weight basis which is produced by a processaccording to claim
 13. 20. An edible textured product, suitable for usein a foodstuff, of which at least 40% of the textured product is fungalcells of the order Mucorales on a dry matter weight basis whichcomprises pellets, granules, a sheet, or is an extrudate, dough, roll,paste or a meat-like chunk which is produced by a process according toclaim
 13. 21. A process for the preparation of a foodstuff comprisingforming a foodstuff with, or adding to an existing, foodstuff, an ediblesubstance according to claim
 5. 22. A process for the preparation of afoodstuff comprising (a) preparing an edible textured product by aprocess comprising mixing one or more, edible component(s) with anedible proteinaceous substance comprising fungal cells of the orderMucorales having a RNA content of below 4 wt % and mechanicallytexturing the mixture, where the proteinaceous product is suitable foruse in a foodstuff, and (b) forming a foodstuff with, or adding to anexisting foodstuff the textured product.
 23. A process for thepreparation of a foodstuff, the process comprising: (i) preparing anedible texture product, by a process comprising mixing one or moreedible proteinaceous substance comprising fungal cells of the orderMucorales having a RNA content of below 4 wt % and mechanicallytexturizing the mixture, wherein the edible proteinaceous substance isprepared by: (a) fermenting fungal cells of the order Mucorales in anaqueous liquid contained in a fermenter vessel, the liquid comprising anassimilable nitrogen (N) source and an assimilable carbon (C) source,and mixing the liquid and cells during fermentation; (b) reducing theRNA content of the fungal cells to below 4% by weight (wt %); (c) beforeor after (b), removing at least some of the water from the mixture offungal cells and aqueous liquid; and (d) processing the fungal cellsinto an edible substance; and (ii) forming a foodstuff with, or addingto an existing foodstuff, the textured product.
 24. A foodstuff whichcomprises an edible product and one or more edible components accordingto claim
 5. 25. A foodstuff which comprises an edible product and one ormore edible components produced by a process according to claim
 15. 26.A foodstuff which comprises an edible product and one or more ediblecomponents produced by a process according to claim
 16. 27. A foodstuffwhich comprises an edible product and one or more edible componentsproduced by a process according to claim
 22. 28. A foodstuff accordingto claim 24 which is a sausage, patty, burger, spread, pâté, animalfeed, tablet, pie, savoury snack, or oven-ready meal.
 29. A foodstuffaccording to claim 25 which is a sausage, patty, burger, spread, pâté,animal feed, tablet, pie, savoury snack, or oven-ready meal.
 30. Afoodstuff according to claim 27 which is a sausage, patty, burger,spread, pâté, animal feed, tablet, pie, savoury snack, or oven-readymeal.